Icam-4 binding sites

ABSTRACT

The present invention relates to intercellular adhesion molecule-4 (ICAM-4), including binding sites on ICAM-4, antagonists affecting ICAM-4 and uses thereof. In one aspect of the invention there is provided an epitope for binding integrins, comprising strands A (or F) and G of domain 1 of ICAM-4. In another aspect of the invention there is provided a footprint domain for binding integrins, comprising a first epitope as defined above and second epitope comprising the C and F strands of domain 1 and the CE loop of domain 2 of ICAM-4.

The present invention relates to intercellular adhesion molecule-4(ICAM-4). In particular, the invention relates to binding sites onICAM-4, antagonists affecting ICAM-4 and uses thereof.

Intercellular adhesion molecule-4 (ICAM-4) is expressed chiefly onerythroid cells and is the glycoprotein that carries the LW blood groupantigens. A study by Bailly et al. (1995, Eur. J. Inumunol. 25:3316-3320) showed binding of integrins LFA-1 and Mac-1 (also known asαMβ2) to ICAM-4.

Another report shows that ICAM-4 binds hemopoietic (HEL) andnon-hemopoietic (FLYRD18, a derivative of HT1080) cell lines and thatthe cellular ligands for ICAM-4 are the α₄β₁ integrin and α_(v)integrins (most notably α_(v)β₁ and α_(v)β₅) respectively (Spring etal., 2001, Blood 98: 458-466).

ICAM-4 possibly has a role in the formation of erythroblastic islands inthe bone marrow (during erythropoiesis) and in the abnormal adhesion ofred cells to activated endothelium in sickle cell disease. There is aneed to understand interactions of ICAM-4 with its receptors for thedevelopment of therapies to diseases in which ICAM-4 is involved. Thesediseases include those involving pathology resulting from abnormaladhesion of red cells to vascular endothelium either directly, orindirectly through binding to other adhesive cells or molecules.Abnormal red cell adhesion is evident in sickle cell disease andmalaria. Red cells from patients with β-thalassaemia major andβ-thalassaemia intermedia also show increased adherence to endotheliumand it has been suggested that this contributes to the microcirculatorydisorders seen in these patients. Red cell-endothelial cell adherencehas also been reported to contribute to the vascular complications foundin diabetes mellitus. Red cell-endothelial cell adherence and red celladherence to other cellular elements in the blood and widerreticuloendothelial system may also be relevant to the pathophysiologyof other conditions where endothelial perturbation or vasculardysfunction occurs; such as strokes, organ transplant rejection,systemic lupus erythematosus and a range of vasculitic and thromboticdisorders. There is preliminary evidence for the involvement of red celladhesion via ICAM-4 in sickle cell disease and deep vein thrombosis.

According to the present invention, there is provided an epitope forbinding integrins, comprising the A and G strands of domain 1 of ICAM-4(SEQ ID NO: 1), in which the A strand (SEQ ID NO: 2) is defined by aminoacid residues 17 to 27 of ICAM-4 and the G strand (SEQ ID NO: 3) isdefined by amino acid residues 90 to 100 of ICAM-4, or a functionalhomologue of the epitope.

The epitope was identified using site-directed mutagenesis of residuesidentified using a molecular model of ICAM-4 derived from the crystalstructure of ICAM-2 (see FIG. 2). The term “ICAM-4” refers herein to themature form of the human protein (as shown in SEQ ID NO: 1), without theN-terminal signal peptide of 30 amino acids found in precursor ICAM-4(see Bailly et al., 1994, Proc. Natl. Acad. Sci. USA91: 5306-5310).Amino acid residues are numbered with reference to this mature ICAM-4.As described in further detail below, our model predicts ICAM-4 i tohave two immunoglobulin superfamily I-set domains, domain 1 beingN-terminal of a membrane-anchored domain 2. According to the model,Domain 1 is an I-1 subset fold with six strands that run in order A, B,C, D, E, F and G. Hence in Domain 1 there is an ABE face and a CDFG(CFG) face. Domain 2 is an I-2 subset fold with seven strands that runin order A, B, C, C′, E, F and G. Hence in Domain 2 there is an ABE anda CC′FG face. Reference to strands herein thus cover both domain 1 ordomain 2 faces.

The epitope of the invention may be defined by amino acid residues F18,W19, V20 on the A strand of ICAM-4 and amino acid residues R92, A94,T95, S96 and R97 on the G strand of ICAM-4.

The epitope of the invention may be modified in that the A strand isreplaced by strand F on domain 1 of ICAM-4, in which the F strand (SEQID NO: 4) is defined by amino acid residues 77 to 87 of ICAM-4. Theepitope here may be defined by amino acid residues W77 and L80 on the Fstrand of ICAM-4 and amino acid residues R92, A94, T95, S96 and R97 onthe G strand of ICAM-4. In the experimental section below, it is shownintegrin ligands of ICAM-4 appear to interact with the A and F strandsof ICAM-4.

Mutagenesis of human ICAM-4 has revealed that modification of theabove-defined single amino acids affect, for example, α_(v)integrin-mediated adhesion to ICAM-4, as elaborated in the experimentalsection below.

The epitope of the invention may be further defined by amino acidresidues W66 on the E strand of domain 1 and K118 on the B strand ofdomain 2 of ICAM-4, in which the E strand (SEQ ID NO: 5) is defined byamino acid residues 160 to 170 of ICAM-4 and the B strand (SEQ ID NO: 6)is defined by amino acid residues 116 to 126 of ICAM-4.

The epitope maybe further defined by amino acid residues N160, V161 andT162 on the E strand of ICAM-4. These residues define an N-glycosylationsite which may have a role in the binding of ICAM-4 and its ligands. Theglycosylation site is located on the top of the E strand (residues160-170) of domain 2 (see FIG. 5). Without an N-glycan chain formed atthe N-glycosylation site, the adhesion between ICAM-4 and its ligands(for example α_(v) ligand) is stronger (see FIG. 4 panels K and L,described below).

Integrins binding to the epitope or part thereof may be α_(v) integrins(for example, as found on HT1080 cells), α₄β1 (also known as VLA-4; forexample, as found on HEL cells and erythroblasts), or α₅β1 (for example,as found on erythroblasts).

In another aspect of the invention, there is provided a footprint domainfor binding integrins, comprising a first epitope as defined above and asecond epitope comprising the C and F strands of domain 1 and the CEloop of domain 2 of ICAM-4, in which the C strand (SEQ ID NO: 7) isdefined by amino acid residues 47 to 54 of ICAM-4, the F strand (SEQ IDNO: 4) is defined as above and the CE loop (SEQ ID NO: 8) is defined byamino acid residues 150 to 158 of ICAM-4, or a functional homologue ofthe footprint domain.

The footprint domain (depicted in FIG. 1 for ICAM-4) can be described asan “adhesive footprint” for multiple integrins. The strands of ICAM-4 asdefined herein arise from their position in a molecular model of ICAM-4that is based on the crystal structure of ICAM-2 (FIG. 2). Evidence isprovided herewith for the involvement of the footprint domain in theinteraction between ICAM-4 and multiple integrin ligands (seeexperimental section below).

The second epitope may be defined by amino acid residues R52 on the Cstrand of ICAM-4, W77 and L80 on the F strand of ICAM-4, T91, W93 andR97 on the G strand of ICAM-4, and E151 and T154 on the CE loop ofICAM-4. This second epitope has been disclosed by Hermand et al. (2000,J. Biol. Chem. 275: 26002-26010).

The integrins binding to the footprint domain or part thereof includeα_(v) integrins (for example, as found on HT1080 cells), VLA-4 (forexample, as found on HEL cells) and/or the β₂-family of integrins (suchas Mac-1, for example, as found on leucocytes and neutrophils, and/orLFA-1), including αLβ2 (for example, as found on neutrophils).

Functional homologues of the epitope or footprint domain includemammalian homologues, for example mouse homologues.

Further provided according to the invention is an antagonist of theepitope and/or the footprint domain as defined herein. For example, theantagonist may be an antibody. Antibodies have the capability todirectly bind to the epitope and/or footprint domain, blocking adhesionto integrin ligands. Antibodies to ICAM-4 have been described by Baillyet al. (1995, Eur. J. Immunol. 25: 3316-3320) and Goel & Diamond (2002,Blood 100: 3797-3803). It is believed that those known antibodies do notbind to the epitope or footprint domain defined herein. If this is notthe case, those known antibodies are excluded from this aspect of theinvention.

Alternatively, an antibody may bind a separate site on ICAM4 and alterthe structural integrity of the epitope and/or footprint domain, therebyreducing affinity and/or inhibiting integrin ligand binding. It isbelieved that the known antibodies to ICAM described by Bailly et al.(1995, supra) and Goel & Diamond (2002, supra) do not alter thestructural integrity of ICAM-4 as described above. If this is not thecase, those antibodies are excluded from this aspect of the invention.

Alternatively, the antagonist of the epitope and/or the footprint domainmay be a compound, for example a low molecular weight compound, whichbinds to the epitope and/or footprint domain to reduce adhesion betweenICAM-4 and its ligands.

In another aspect of the invention there is provided an antagonist of aligand for the epitope and/or the footprint domain defined herein. Theantagonist may have or consist essentially of three, four, five, six,seven, eight, nine or more amino acid residues of the A, C, F or Gstrands or the CE loop of ICAM-4 or a functional homologue thereof. Forexample, the antagonist of a ligand for the epitope and/or the footprintdomain may have or consist essentially of the amino acid sequenceaccording to SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Theantagonist may comprise an active site having or consisting essentiallyof the amino acid sequence according to SEQ ID NO: 9, SEQ ID NO: 10 orSEQ ID NO: 11.

Experimental evidence (below) demonstrates inhibition of binding betweenICAM-4 and various ligands (such as integrins).

Alternatively, the antagonist of a ligand for the epitope and/or thefootprint domain may comprise other peptides, drugs or antibodies whichbind to the ligand and thus reduce adhesion of the ligand to the epitopeand/or the footprint domain

In a further aspect of the invention, there is provided a method ofantagonising the epitope and/or the footprint domain, comprising thestep of contacting the epitope and/or the footprint domain with theantagonist to the epitope and/or the footprint domain described herein.There is also provided a method of antagonising a ligand of the epitopeand/or the footprint domain, comprising the step of contacting theligand (or an environment such as a solution containing the ligand) withthe antagonist of the ligand described herein. Our data shows that suchantagonists (for example SEQ ID NOs: 9, 10 or 11) effective blockbinding to ICAM4.

Another aspect of the invention is the use of the antagonist as definedherein for treating a disease, for example a disease involving ICAM-4.Furthermore, the invention covers use of an antagonist as describedherein in the manufacture of a medicament for the treatment of a diseaseinvolving ICAM-4. The disease may be characterised by increased ordecreased levels of ICAM4 binding compared with ICAM-4 binding inhealthy individuals.

We have found that the above epitope and footprint domain mediateadhesion to several integrins, and if this adhesion is blocked, forexample, therapeutic effects may be possible in diseases such as sicklecell disease, deep vein thrombosis (DVT), malaria, strokes and moregenerally, vascular complications in any other condition found inmammals (heart disease, diabetes, β-thalassaemia, thromboticcomplications of haematological diseases) may be possible. For example,in sickle cell disease it is thought that ICAM-4 binds sickle red cellsto the endothelium. This abnormal binding may be prevented using anantagonist of ICAM-4.

In a further aspect there is provided an isolated nucleotide encodingthe epitope or the footprint domain or the antagonist defined herein.For example, the isolated nucleotide encoding the epitope or thefootprint domain or the antagonist may have a sequence defined withinthe sequence of SEQ ID NO: 12.

Embodiments of the invention will be described hereafter with referenceto the accompanying figures, of which:

FIG. 1 shows a molecular model of ICAM4 depicting the entire footprintdomain;

FIG. 2 shows a molecular model of ICAM-4 depicting the ABE faces and theCFG faces;

FIG. 3 shows a molecular model of the α₄β₁ and α_(v) integrin bindingdomain of ICAM-4;

FIG. 4 shows graphs A-L depicting the effect of mutating single residuesof human ICAM-4Fc on the adhesion of HT1080 cells (exhibiting α_(v)integrin);

FIG. 5 shows a molecular model of the ICAM-4 N-glycosylation site indomain 2;

FIG. 6 shows a molecular model of the LFA-1 and Mac-i binding footprintof ICAM-4;

FIG. 7 is a histogram showing human ICAM-4 peptide inhibition of HELcell binding to human ICAM-4Fc coated at 5 μg/ml;

FIG. 8 is a histogram showing the results of FIG. 7 as a percentage ofbinding to human ICAM-4Fc in the absence of peptides;

FIG. 9 is a histogram showing human ICAM-4 peptide inhibition of HT1080cell binding to human ICAM-4Fc coated at 7.5 μg/ml;

FIG. 10 is a histogram showing the results of FIG. 8 as a percentage ofbinding to human ICAM-4Fc in the absence of peptides;

FIG. 11 is a histogram showing human ICAM-4 peptide inhibition of HELcell binding to murine ICAM-4Fc coated at 5 μg/ml;

FIG. 12 is a histogram showing the results of FIG. 11 as a percentage ofbinding to murine ICAM-4Fc in the absence of peptides;

FIG. 13 is a histogram showing human ICAM-4 peptide inhibition of HT1080cell binding to murine ICAM-4Fc coated at 51 μg/ml;

FIG. 14 is a histogram showing the results of FIG. 13 as a percentage ofbinding to murine ICAM-4Fc in the absence of peptides;

FIG. 15 is a histogram showing human ICAM-4 peptide inhibition of HELcell binding to human ICAM-4Fc coated at 2.5 μg/ml;

FIG. 16 is a histogram showing the results of FIG. 15 as a percentage ofbinding to ICAM-4Fc in the absence of peptides;

FIG. 17 is a histogram showing human ICAM-4 peptide inhibition of HT1080cell binding to human ICAM-4Fc coated at 5 μg/ml;

FIG. 18 is a histogram showing the results of FIG. 17 as a percentage ofbinding to ICAM-4Fc in the absence of peptides;

FIG. 19 is a histogram showing that ICAM-4 peptides block adhesionbetween erythroblasts and ICAM-4; and

FIG. 20 is a histogram showing that blocking beta 2 antibody and ICAM-4peptides inhibit adhesion between neutrophils and ICAM4.

The figure legends in more detail are:

FIG. 1 Molecular model of ICAM-4 with the entire “footprint” (The A, Gand F strand of domain 1, extending down towards the CE loop of domain2), along with residues W66 and K118 are shown in grey. Views A, B and Care rotated 120° with respect to each other.

FIG. 2. Molecular model of ICAM4 with the ABE faces shaded grey and theCFG faces are un-shaded. Views A and B are rotated 180° with respect toeach other. Domain 1 is at the top of the model and is highlighted by aand domain 2 at the bottom of the model is highlighted by b.

FIG. 3. The α₄β₁ and α_(v) integrin binding footprint of ICAM-4. Views Aand B are rotated 180° with respect to each other. The mutated residuesthat comprise the α₄β₁ and α_(v) integrin binding footprint in the Astrand are in light grey (a), and those in the G strand are in dark grey(b). Dark grey residues in the E strand of domain 1 (W66, c) and Bstrand of domain 2 (K 18, d) also affect α₄β₁ and a_(v) integrinbinding.

FIG. 4. The effect of mutating single residues of human ICAM-4Fc on theadhesion of HT1080 cells. x-axis: wild-type and mutant human ICAM-4Fccoating concentration (μg/ml); y-axis: percentage of input cells bound.Triangles show titrations of wild-type ICAM-4Fc and diamonds showtitrations of mutant ICAM-4Fc. A, F18A mutant; B, W19A mutant; C, V20Tmutant; D, R92E mutant; E, A94L mutant; F, T95V mutant; G, S96A mutant;H, R97E mutant; I, W66A mutant; J, K118E mutant; K, N160A mutant; L,T162V mutant. Results shown are representative (one of several repeatexperiments). Results are shown as mean (n=3)±1 standard deviation.

FIG. 5. The ICAM-4 N-glycosylation site in domain 2. Views A and B arerotated 180° with respect to each other. Residues N160 (a) and T162 (b)are highlighted in dark grey.

FIG. 6. The LFA-1 and Mac-1 binding footprint of ICAM-4. Views A and Bare rotated 180° with respect to each other. View A shows the Mac-1footprint with domain 1 residues in the C, F and G strands highlightedin dark grey and domain 2 residues in the C′ E loop highlighted in lightgrey. View B shows the LFA-1 footprint with the domain 1 residues in theF and G strands highlighted in dark grey.

FIG. 7. Human ICAM-4 peptide inhibition of HEL cell binding to humanICAM-4Fc. x-axis: binding of HEL cells in the presence of assay buffer,defined peptides or EDTA; y-axis: percentage of input cells bound. a-hshows binding to human ICAM-4Fc and i shows binding to human NCAMFc. a,assay buffer; b, svpFWVrms peptide (SEQ ID NO: 9); c, tRwATSRit peptide(SEQ ID NO: 10), d, rqgktlrgp peptide (SEQ ID NO: 13); e, svpFWVrmspeptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQ ID NO: 13); f,tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgp peptide (SEQ ID NO:13); g, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRit peptide (SEQ IDNO: 10); h, EDTA; i, assay buffer. Human ICAM-4Fc was coated at aconcentration of 5 μg/ml, peptides were used at 500 μM finalconcentration for each peptide, and each data point is the mean of threeindependent assays.

FIG. 8. Human ICAM-4 peptide inhibition of HEL cell binding to humanICAM-4Fc. x-axis: binding of HEL cells in the presence of assay buffer,defined peptides or EDTA; y-axis: input cells bound expressed as apercentage of binding to ICAM-4Fc in the absence of peptides. a-h showsbinding to human ICAM-4Fc and i shows binding to human NCAMFc. a, assaybuffer (100%); b, svpFWVrms peptide (SEQ ID NO: 9) (61%); c, tRwATSRitpeptide (SEQ ID NO: 10) (58%); d, rqgktlrgp peptide (SEQ ID NO: 13)(107%); e, svpFWVrms peptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQID NO: 13) (60%); f, tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgppeptide (SEQ ID NO: 13) (56%); g, svpFWVrms peptide (SEQ ID NO: 9) plustRwATSRit peptide (SEQ ID NO: 10) (35%/0); h, EDTA (1%); i, assay buffer(3%). Human ICAM-4Fc was coated at a concentration of 5 μg/ml, peptideswere used at 500 μM final concentration for each peptide, and each datapoint is the mean of three independent assays.

FIG. 9. Human ICAM-4 peptide inhibition of HT1080 cell binding to humanICAM-4Fc. x-axis: binding of HT1080 cells in the presence of assaybuffer, defined peptides or EDTA; y-axis: percentage of input cellsbound. a-h shows binding to human ICAM-4Fc and i shows binding to humanNCAMFc. a, assay buffer; b, svpFWVrms peptide (SEQ ID NO: 9); c,tRwATSRit peptide (SEQ ID NO: 10), d, rqgktlrgp peptide (SEQ ID NO: 13);e, svpFWVrms peptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQ ID NO:13); f, tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgp peptide (SEQ IDNO: 13); g, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRit peptide (SEQID NO: 10); h, EDTA; i, assay buffer. Human ICAM-4Fc was coated at aconcentration of 7.5 μg/ml, peptides were used at 500 μM finalconcentration for each peptide, and each data point is the mean of threeindependent assays.

FIG. 10. Human ICAM-4 peptide inhibition of HT1080 cell binding to humanICAM-4Fc. x-axis: binding of HT1080 cells in the presence of assaybuffer, defined peptides or EDTA; y-axis: input cells bound expressed asa percentage of binding to human ICAM4Fc in the absence of peptides. a-hshows binding to human ICAM-4Fc and i shows binding to human NCAMFc. a,assay buffer; b, svpFVWVrms peptide (SEQ ID NO: 9) (46%); c, tRwATSRitpeptide (SEQ ID NO: 10) (60%); d, rqgktkgp peptide (SEQ ID NO: 13)(97%); e, svpFWVrms peptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQID NO: 13) (37%); f, tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgppeptide (SEQ ID NO: 13) (44%); g, svpFWVrms peptide (SEQ ID NO: 9) plustRwATSRit peptide (SEQ ID NO: 10) (32%); h, EDTA (6%); i, assay buffer(5%). Human ICAM-4Fc was coated at a concentration of 7.5 μg/ml,peptides were used at 500 μM final concentration for each peptide, andeach data point is the mean of three independent assays.

FIG. 11. Human ICAM-4 peptide inhibition of HEL cell binding to murineICAM-4Fc. x-axis: binding of HEL cells in the presence of assay buffer,defined peptides or EDTA; y-axis: percentage of input cells bound. a-hshows binding to murine ICAM-4Fc and i shows binding to human NCAMFc. a,assay buffer; b, svpFWVrms peptide. (SEQ ID NO: 9); c, tRwATSRit peptide(SEQ ID NO: 10), d, rqgktlrgp peptide (SEQ ID NO: 13); e, svpFWVrmspeptide (SEQ ID NO: 9) plus rqgkllrgp peptide (SEQ ID NO: 13); f,tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgp peptide (SEQ ID NO:13); g, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRit peptide (SEQ IDNO: 10); h, EDTA; i, assay buffer. Murine ICAM-4Fc was coated at aconcentration of 5 μg/ml, peptides were used at 500 μM finalconcentration for each peptide, and each data point is the mean of twoindependent assays.

FIG. 12. Human ICAM-4 peptide inhibition of HEL cell binding to murineICAM-4Fc. x-axis: binding of HEL cells in the presence of assay buffer,defined peptides or EDTA; y-axis: input cells bound expressed as apercentage of binding to murine ICAM-4Fc in the absence of peptides. a-hshows binding to murine ICAM-4Fc and i shows binding to human NCAMFc. a,assay buffer; b, svpFWVrms peptide (SEQ ID NO: 9) (67%); c, tRwATSRitpeptide (SEQ ID NO: 10) (58%); d, rqgktlrgp peptide (SEQ ID NO: 13)(94%); e, svpFWVrms peptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQID NO: 13) (70%); f, tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgppeptide (SEQ ID NO: 13) (55%); g, svpFWVrms peptide (SEQ ID NO: 9) plustRwATSRit peptide (SEQ ID NO: 10) (47%); h, EDTA (8%); i, assay buffer(19%). Murine ICAM-4Fc was coated at a concentration of 5 μg/ml,peptides were used at 500 μM final concentration for each peptide, andeach data point is the mean of two independent assays.

FIG. 13. Human ICAM-4 peptide inhibition of HT1080 cell binding tomurine ICAM-4Fc. x-axis: binding of HT1080 cells in the presence ofassay buffer, defined peptides or EDTA; y-axis: percentage of inputcells bound. a-h shows binding to murine ICAM-4Fc and i shows binding tohuman NCAMFc. a, assay buffer; b, svpFWVrms peptide (SEQ ID NO: 9); c,tRwATSRit peptide (SEQ ID NO: 10), d, rqgktlrgp peptide (SEQ ID NO: 13);e, svpFWVrms peptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQ ID NO:13); f, tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgp peptide (SEQ IDNO: 13); g, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRit peptide (SEQID NO: 10); h, EDTA; i, assay buffer. Murine ICAM-4Fc was coated at aconcentration of 5 μg/ml, peptides were used at 500 μM finalconcentration for each peptide, and each data point is the mean of twoindependent assays.

FIG. 14. Human ICAM-4 peptide inhibition of HT1080 cell binding tomurine ICAM-4Fc. x-axis: binding of HT1080 cells in the presence ofassay buffer, defined peptides or EDTA; y-axis: input cells boundexpressed as a percentage of binding to murine ICAM-4Fc in the absenceof peptides. a-h shows binding to murine ICAM-4Fc and i shows binding tohuman NCAMFc. a, assay buffer; b, svpFWVrms peptide (SEQ ID NO: 9)(60%); c, tRwATSRit peptide (SEQ ID NO: 10) (80%); d, rqgktlrgp peptide(SEQ ID NO: 13) (92%); e, svpFWVrms peptide (SEQ ID NO: 9) plusrqgktlrgp peptide (SEQ ID NO: 13) (61%); f, tRwATSRit peptide (SEQ IDNO: 10) plus rqgktlrgp peptide (SEQ ID NO: 13) (74%); g, svpFWVrmspeptide (SEQ ID NO: 9) plus tRwATSRit peptide (SEQ ID NO: 10) (51%); h,EDTA (1%); i, assay buffer (1%). Murine ICAM-4Fc was coated at aconcentration of 5 μg/ml, peptides were used at 500 μM finalconcentration for each peptide, and each data point is the mean of twoindependent assays.

FIG. 15. Human ICAM-4 peptide inhibitions of HEL cell binding to humanICAM-4Fc. x-axis: binding of HEL cells in the presence of assay buffer,defined peptides or EDTA, y-axis: percentage of input cells bound. a-pshows binding to human ICAM-4Fc. a, assay buffer, b, assay buffer plus 2mM EDTA c svpFWVrms peptide (SEQ ID NO: 9), d, tRwATSRit peptide (SEQ IDNO: 10), e, aWssLahcl peptide (SEQ ID NO: 11), f, rqgktlrgp peptide (SEQID NO: 13), g, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRit peptide(SEQ ID NO: 10), h, svpFWVrms peptide (SEQ ID NO: 9) plus aWssLahclpeptide (SEQ ID NO: 11), i, tRwATSRit peptide (SEQ ID NO: 10) plusaWssLahcl peptide (SEQ ID NO: 11), j, svpFWVrms peptide (SEQ ID NO: 9)plus rqgktlrgp peptide (SEQ ID NO: 13), k, tRwATSRit peptide (SEQ ID NO:10) plus rqgktlrgp peptide (SEQ ID NO: 13), 1, aWssLahcl peptide (SEQ IDNO: 11) plus rqgktlrgp peptide (SEQ ID NO: 13), m, svpFWVrms peptide(SEQ ID NO: 9) plus tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgpeptide (SEQ ID NO: 13), n, svpFWVrms peptide (SEQ ID NO: 9) plusaWssLahcl peptide (SEQ ID NO: 11) plus rqgktlrgp peptide (SEQ ID NO:13), o, tRwATSRit peptide (SEQ ID NO: 10) plus aWssLahcl peptide (SEQ IDNO: 11) plus rqgktlrgp peptide (SEQ ID NO: 13), p, svpFWVrms peptide(SEQ ID NO: 9) plus tRwATSRit peptide (SEQ ID NO: 10) plus aWssLahclpeptide (SEQ ID NO: 11). Human ICAM-4Fc was coated at a concentration of2.5 μg/ml, peptides were used at 750 μM final concentration for eachpeptide, and each data point is the mean of two independent assays

FIG. 16. Human ICAM-4 peptide inhibitions of HEL cell binding to humanICAM-4Fc. x-axis: binding of HEL cells in the presence of assay buffer,defined peptides or EDTA, y-axis: input cells bound expressed as apercentage of binding to human ICAM-4Fc in the absence of peptides. a,assay buffer; b, assay buffer plus 2 mM EDTA (26%); c, svpFWVrms peptide(SEQ ID NO: 9) (64%); d, tRwATSRit peptide (SEQ ID NO: 10) (58%); e,aWssLahcl peptide (SEQ ID NO: 11) (50%/o); f, rqgktlrgp peptide (SEQ IDNO: 13) (105%); g, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRitpeptide (SEQ ID NO: 10) (52%); h, svpFWVrms peptide (SEQ ID NO: 9) plusaWssLahcl peptide (SEQ ID NO: 11) (43%); i, tRwATSRit peptide (SEQ IDNO: 10) plus aWssLahcl peptide (SEQ ID NO: 11) (41%); j, svpFWVrmspeptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQ ID NO: 13) (59%); k,tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgp peptide (SEQ ID NO: 13)(55%); 1, aWssLahcl peptide (SEQ ID NO: 11) plus rqgktlrgp peptide (SEQID NO: 13) (46%); m, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRitpeptide (SEQ ID NO: 10) plus rqgktlrg peptide (49%/o); n, svpFWVrmspeptide (SEQ ID NO: 9) plus aWssLahcl peptide (SEQ ID NO: 11) plusrqgktlrgp peptide (SEQ ID NO: 13) (42%); o, tRwATSRit peptide (SEQ IDNO: 10) plus aWssLahcl peptide (SEQ ID NO: 11) plus rqgktlrgp peptide(SEQ ID NO: 13) (40%); p, svpFWVrms peptide (SEQ ID NO: 9) plustRwATSRit peptide (SEQ ID NO: 10) plus aWssLahcl peptide (SEQ ID NO: 11)(42%). Human ICAM-4Fc was coated at a concentration of 2.5 μg/ml,peptides were used at 750 μM final concentration for each peptide, andeach data point is the mean of two independent assays.

FIG. 17. Human ICAM-4 peptide inhibitions of HT1080 cell binding tohuman ICAM-4Fc. x-axis: binding of HT1080 cells in the presence of assaybuffer, defined peptides or EDTA, y-axis: percentage of input cellsbound. a-p shows binding to human ICAM-4Fc. a, assay buffer, b, assaybuffer plus 2 mM EDTA c svpFWVrms peptide (SEQ ID NO: 9), d, tRwATSRitpeptide (SEQ ID NO: 10), e, aWssLahcl peptide (SEQ ID NO: 11), f,rqgktlrgp peptide (SEQ ID NO: 13), g, svpFWVrms peptide (SEQ ID NO: 9)plus tRwATSRit peptide (SEQ ID NO: 10), h, svpFWVrms peptide (SEQ ID NO:9) plus aWssLahcl peptide (SEQ ID NO: 11), i, tRwATSRit peptide (SEQ IDNO: 10) plus aWssLahcl peptide (SEQ ID NO: 11), j, svpFWVrms peptide(SEQ ID NO: 9) plus rqgktlrgp peptide (SEQ ID NO: 13), k, tRwATSRitpeptide (SEQ ID NO: 10) plus rqgktlrgp peptide (SEQ ID NO: 13), 1,aWssLahcl peptide (SEQ ID NO: 11) plus rqgktlrgp peptide (SEQ ID NO:13), m, svpFWVrms peptide (SEQ ID NO: 9) plus tRwATSRit peptide (SEQ IDNO: 10) plus rqgktlrg peptide, n, svpFWVrms peptide (SEQ ID NO: 9) plusaWssLahcl peptide (SEQ ID NO: 11) plus rqgktlrgp peptide (SEQ ID NO:13), o, tRwATSRit peptide (SEQ ID NO: 10) plus aWssLahcl peptide (SEQ IDNO: 11) plus rqgktlrgp peptide (SEQ ID NO: 13), p, svpFWVrms peptide(SEQ ID NO: 9) plus tRwATSRit peptide (SEQ ID NO: 10) plus aWssLahclpeptide (SEQ ID NO: 11). Human ICAM-4Fc was coated at a concentration of5 μg/ml, peptides were used at 750 μM final concentration for eachpeptide, and each data point is the mean of two independent assays.

FIG. 18. Human ICAM-4 peptide inhibitions of HT1080 cell binding tohuman ICAM-4Fc. x-axis: binding of HT1080 cells in the presence of assaybuffer, defined peptides or EDTA, y-axis: input cells bound expressed asa percentage of binding to human ICAM-4Fc in the absence of peptides. a,assay buffer; b, assay buffer plus 2 mM EDTA (10%); c, svpFWVrms peptide(SEQ ID NO: 9) (41%); d, tRwATSRit peptide (SEQ ID NO: 10) (42%); e,aWssLahcl peptide (SEQ ID NO: 11) (71%); f, rqgktlrgp peptide (SEQ IDNO: 13) (96%); g, svpFWVrVns peptide (SEQ ID NO: 9) plus tRwATSRitpeptide (SEQ ID NO: 10) (46%); h, svpFWVnns peptide (SEQ ID NO: 9) plusaWssLahcl peptide (SEQ ID NO: 11) (52%); i, tRwATSRit peptide (SEQ IDNO: 10) plus aWssLahcl peptide (SEQ ID NO: 11) (50%); j, svpFWVrmspeptide (SEQ ID NO: 9) plus rqgktlrgp peptide (SEQ ID NO: 13) (40%/o);k, tRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrgp peptide (SEQ ID NO:13) (39%); 1, aWssLahcl peptide (SEQ ID NO: 11) plus rqgktlrgp peptide(SEQ ID NO: 13) (64%); m, svpFWVrms peptide (SEQ ID NO: 9) plustRwATSRit peptide (SEQ ID NO: 10) plus rqgktlrg peptide (39%); n,svpFWVrms peptide (SEQ ID NO: 9) plus aWssLahcl peptide (SEQ ID NO: 11)plus rqgktlrgp peptide (SEQ ID NO: 13) (50%); o, tRwATSRit peptide (SEQID NO: 10) plus aWssLahcl peptide (SEQ ID NO: 11) plus rqgktlrgp peptide(SEQ ID NO: 13) (48%); p, svpFWVrms peptide (SEQ ID NO: 9) plustRwATSRit peptide (SEQ ID NO: 10) plus aWssLahcl peptide (SEQ ID NO: 11)(52%). Human ICAM-4Fc was coated at a concentration of 5 μg/ml, peptideswere used at 750 μM final concentration for each peptide, and each datapoint is the mean of two independent assays.

FIG. 19. ICAM-4 peptides block adhesion between erythroblasts andICAM-4. Erythroblast adhesion to 5 μg/ml ICAM-4 was performed in thepresence of 5 μg/ml of the beta 1 integrin activating antibody TS2/16.Peptides were used at 500 μM. Results are expressed as a % of theerythroblasts bound to ICAM4 minus those bound to NCAM in the presenceof the rqgktlrgp peptide (SEQ ID NO: 13) (i.e. % control cells bound).a, svpFWVrms peptide (SEQ ID NO: 9); b, tRwATSRit peptide (SEQ ID NO:10); c, aWssLahcl peptide (SEQ ID NO: 11); d, rqgktlrgp peptide (SEQ IDNO: 13).

FIG. 20. Blocking beta 2 antibody and ICAM-4 peptides inhibit adhesionbetween neutrophils and ICAM-4. Neutrophil adhesion to 2.5 μg/ml ICAM-4was performed in the presence of 10 μg/ml of the integrin blockingantibodies and 500 μM of the peptides. Results are expressed as a % ofthe neutrophils bound to ICAM-4 in the absence of either antibody orpeptide (i.e. % control cells bound). a, beta 1; b, beta 2; c, beta 3;d, svpFWVrms peptide (SEQ ID NO: 9); e, tRwATSRit peptide (SEQ ID NO:10); f, aWssLahcl peptide (SEQ ID NO: 11).

Experimental

In order to elucidate the structural basis of integrin-ICAM-4interaction, in Example 1 we analysed surface-exposed residues, bysite-directed mutagenesis, using a molecular model of ICAM-4 derivedfrom the crystal structure of ICAM-2. The model presents ICAM-4 as twoIg-like domains; domain 1 being N-terminal of the membrane anchoreddomain 2. Each domain has two faces (or sides); the ABE and the CC′FGfaces (FIG. 2). Mutagenesis of ICAM-4 has revealed that a number ofsingle amino acid changes affect α_(v) integrin-mediated adhesion toICAM-4. Peptide inhibition data confirms the mutagenesis data andprovides evidence that the same footprint is relevant to ICAM-4'sinteraction with α₄β₁. Due to the overlap of the α_(v) integrin and a4p,binding site with that of the binding site of LFA-1 and Mac-1 we predictthat the peptides also inhibit any ICAM4/LFA-1 or Mac-1 interaction. InExamples 2 and 3, we show that blocking peptides (antagonists) arecapable of inhibiting erythroblast and neutrophil adhesion to ICAM-4,suggesting that such antagonists can be useful in treating diseasesrelating to ICAM-4 dysfunction.

EXAMPLE 1

Cell Adhesion Assay

Cell adhesion assays were performed as described in Spring et al. 2001(supra). Immulon4 96 well plates (Dynex Technologies, Billingshurst,United Kingdom) were coated with 1 μg/well goat-antihuman-Fc (Sigma,Poole, United Kingdom) for 24 hours at 4° C., washed three times withPBS and coated with an Fc fusion ICAM-4 protein for 18 hours at 4° C.before blocking with 0.4% BSA PBS for 2 hours at 22° C. Cells werelabelled with 10 μg/ml 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein acetoxymethyl ester in assay buffer (IMEM, 2 mM EGTA,10 μg/ml human ivIgG) for 15 minutes at 37° C. HT1080 cells wereactivated with 80 μM phorbol myristate acetate prior to both cells beingwashed with assay buffer containing 2 mM Mn²⁺. Cells, at 5×10⁴ cells perwell, were added to the ICAM-4Fc-coated plates for 30 minutes at 37° C.,prior to being given repeated washes in assay buffer and read on afluorescence microplate reader (excitation 485 nm, emission 530 nm). Thepercentage of bound cells was calculated after each wash. Peptideinhibition was performed by incubating the cells with 500 μM peptide at0° C. for 15 minutes ahead of their addition, still in the presence of500 μM peptide to the ICAM-4Fc coated plates. In peptide inhibitionstudies the appropriate ICAM-4Fc coating concentration for each cellline was pre-determined by titration of ICAM-4Fc and the lowestconcentration at which maximal binding was achieved was used to coat theplates

Preparation of ICAM-4Fc Fusion Proteins

Point mutations were inserted into ICAM-4 in pIg vector (see Simmons DL, 1993, Cloning cell surface molecules by transient expression inmammalian cells, In: Hartley D A, ed. Cellular interactions indevelopment. New York, N.Y.:IRL press, 93-127) by PCR amplification overtwo stages. Oligonucleotides (see “Mutagenesis primers” below)containing mismatched bases, together with 5′-agaacccactgcttactggct (SEQID NO: 14) and 3′-tgagcctgcttccagcagca (SEQ ID NO: 15) primers were usedto generate two overlapping products. Following gel purification the twooverlapping PCR products were annealed together before finalamplification using the 5′ and 3′ primers. The final PCR product wasrestricted and ligated into pIg vector. All mutant clones were verifiedby sequence analysis. Mutant ICAM-4Fc proteins were expressed in COS-7cells as described in Simmons D L (1993, supra), and purified fromculture supernatant on protein A-Sepharose. TABLE 1 Mutagenesis primers(all shown in 5′-3′ orientation) F18A tca gtg ccc GCc tgg gtg cgc (SEQID NO:16) gcg cac cca gGC ggg cac tga (SEQ ID NO:17) W19A gtg ccc ttcGCg gtg cgc atg (SEQ ID NO:18) cat gcg cac cGC gaa ggg cac (SEQ IDNO:19) V20T ccc ttc tgg ACg cgc atg agc (SEQ ID NO:20) gct cat gcg cGTcca gaa ggg (SEQ ID NO:21) R92E gga aaa aca GAA tgg gcc ac (SEQ IDNO:22) gt ggc cca TTC tgt ttt tcc (SEQ ID NO:23) A94L aca cgc tgg CTcacc tcc agg (SEQ ID NO:24) cct gga ggt gAG cca gcg tgt (SEQ ID NO:25)T95V cgc tgg gcc GTc tcc agg at (SEQ ID NO:26) at cct gga gAC ggc ccagcg (SEQ ID NO:27) S96A tgg gcc acc Gcc agg atc acc (SEQ ID NO:28) ggtgat cct ggC ggt ggc cca (SEQ ID NO:29) R97E gcc acc tcc GAg atc acc gc(SEQ ID NO:30) gc ggt gat cTG gga ggt ggc (SEQ ID NO:31) W66A ggg ccgggt GCg gtg tct ta (SEQ ID NO:32) ta aga cac cGC acc cgg ccc (SEQ IDNO:33) K118E aag ggc agg Gaa tac act tt (SEQ ID NO:34) aa agt gta ttCcct gcc ctt (SEQ ID NO:35) N160A gat ctg gcc GCc gtg acc ttg (SEQ IDNO:36) caa ggt cac gGC ggc cag atc (SEQ ID NO:37) T162V gcc aac gtg GTcttg acc ta (SEQ ID NO:38) ta ggt caa gAC cac gtt ggc (SEQ ID NO:39)Results and Discussion

ICAM-4 is predicted to have two immunoglobulin superfamily I-setdomains, domain 1 being N-terminal of the membrane anchored domain 2. Ona molecular model of ICAM-4 (Spring et al. 2001, supra, and see FIG. 2),based on the crystal structure of ICAM-2, the VLA-4 and α_(v) integrinbinding epitope on ICAM-4 consists of eight residues and is located indomain 1, in between the ABE and CFG faces (FIG. 3). Three of theresidues, F18, W19, and V20, are positioned on the A strand (FIG. 3 a),and five residues, R92, A94, T95, S96 and R97 are on the G strand (FIG.3 b). Each of these residues was identified as important for binding onthe basis of a decrease in binding of the singly mutated ICAM-4 and theα_(v) integrin ligand (see FIG. 4 panels A through L). These residuesidentify an epitope on the ICAM-4 molecule that straddles the edges ofboth the ABE and CC′FG face of domain 1.

There is also a published LFA-1/Mac-1 binding site (Hermand et al.,2000, supra) on ICAM-4 which is comprised of 8 residues, T91, R52, E151,T154, W93, L80, R97 and W77. On domain 1, T91, W93 and R97 are on the Gstrand (residues 90-100), W77 and L80 are on the F strand (residues77-87) and R52 is on the C strand (residues 47-54). On domain 2 E151 andT154 are on the C′-E loop (residues 150-158). Of these residues, allcomprise the Mac-1 binding site (FIG. 6 view A) however, W93, L80, R97and W77 only comprise the LFA-1 binding site (FIG. 6 view B).

In total, the footprint domain of the present invention comprises awider area than that covered by the epitope defined by the residuesmutated herein (see FIG. 4). It comprises these residues, the residuesdescribed by Hermand et al. (2000, supra) and amino-acids in thesurrounding area. The footprint comprises residues on the A, C, G and Fstrand of domain 1 and extends down to the CE loop in domain 2 (seeFIGS. 1, 2, 3, 5, and 6).

Two other residues are thought to be involved in the interaction betweenICAM-4 and its integrin ligands; W66, located on the E strand (residues65-75) of domain 1 and K118, which is found on the B strand (residues116-126) of domain 2 (FIG. 3 view A and residues c and d, respectivelyand FIG. 4 panels I and J respectively). These mutations also decreasethe level of adhesion between ICAM-4 and HT1080 cells.

In addition, an N-glycosylation site comprising residues N160, V161 andT162 is believed to have a role in the binding of ICAM-4 and itsligands. This site is located at the top of the E strand (residues160-170) of domain 2 (see FIG. 5 views A and B; N160 is arrowed by a,and T162 is arrowed by b). Mutation of N160 or T162 leads to an elevatedlevel of adhesion between ICAM-4 and HT1080 cells (FIG. 4 panels K andL). Analysis by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis revealed that the N160A and T162V mutants have increasedelectrophoretic mobility than native ICAM-4, which suggests that thesetwo “super adhesive” mutations prevent the N-glycosylation of asparagine160.

Areas thought to be important in ICAM-4 binding are shown in FIGS. 1, 3,5 and 6.

FIGS. 7-18 show inhibition of HEL cell binding and HT1080 cell bindingto human and murine ICAM-4Fc in the presence of blocking peptidesequences (peptides svpFWVrms (SEQ ID NO: 9), tRwATSRit (SEQ ID NO: 10)and aWssLahcl (SEQ ID NO: 11)) and a control peptide (rqgktlrgp; SEQ IDNO: 13).

Our findings suggest that contact between ICAM-4 and its integrinligands involves a large extent of the surface of ICAM-4, with theepitope on domain 1 being a critical site in mediating this interaction.Integrin-mediated adhesion to ICAM-4 may play a role in the formation oferythroblastic islands in the bone marrow (during erythropoiesis) and inthe abnormal adhesion of red cells to activated endothelium and othercellular elements in the vasculature and wider reticuloendothelialsystem in the diseases mention above.

EXAMPLE 2

Peptide Inhibition of Erythroblast Adhesion to ICAM-4

In Example 1, we identified an area on ICAM-4 that is important in itsadhesion to αV integrins and using this information we designed blockingpeptides corresponding to the sequences of the A, D, F and G strands ofdomain 1. These peptides have the sequences S(15)VPFWVRMS (SEQ ID NO: 9;on A strand), R(56)QGKTLRGP (SEQ ID NO: 13; on D strand), A(76)WSSLAHCL(SEQ ID NO: 11; on F Strand) and T(91)RWATSRIT (SEQ ID NO: 10; on Gstrand). We have shown that early erythroblasts bind to ICAM-4 in thepresence of TS2/16, an activating β1 antibody (unpublishedobservations). The adhesion of HEL cells to ICAM-4 is mediated by theα4β1 integrin but not the α5β1 integrin (Spring et al., 2001, supra).Erythroblasts express only two integrins at this stage indifferentiation: α4β1 and α5β1 (unpublished observations). Therefore wehypothesise that erythroblasts adhere to ICAM-4 via α4β1, although wehave not ruled out the fact that α5β1 may be involved in thisinteraction.

We have utilised the blocking ICAM-4 peptides (i.e., SEQ ID NOs: 9, 10,11 and 13—see Example 1) in order to inhibit the adhesion of day 4erythroblasts to ICAM-4 (see FIG. 19). Erythroblast cultures wereinitiated from CD34 positive cells purified from pooled buffy coatresidues (obtained from the National Blood Service, Bristol, UK) andmaintained as described in Southcott et al. (1999, Blood 93: 4425-4435).Cell adhesion assays were performed as described in Example 1 above.Immulon-4 96 well plates (Dynes Technologies, Billingshurst, UK) werecoated with 1 μg/well goat-antihuman-Fc (Sigma, Poole, UK) for 24 hoursat 4° C., washed three times with PBS and coated with 0.25 μg/well Fcfusion ICAM-4 protein (ICAM-4Fc) for 18 hours at 4° C. before blockingwith 0.4% BSA PBS for 2 hours at 22° C. Erythroblasts were labelled with10 μg/ml 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluoresceinacetoxymethyl ester (Sigma, Poole, UK) in assay buffer (Iscoves modifiedEagle medium, 2 mM EGTA, 0.1% BSA, 10 μg/ml Immune globulin intravenous(human) (Cutter Biological, Newbury, Berks, UK)) for 15 minutes at 37°C. Erythroblasts were washed with assay buffer containing 2 mM Mn²⁺.Erythroblasts, at 5×10⁴ cells per well, were added to theICAM-4Fc-coated plates for 30 minutes at 37° C., prior to beingcyclically read on a fluorescence microplate reader (excitation 485 nm,emission 530 nm) and washed in assay buffer. The percentage of boundcells was calculated after each wash Peptide inhibition was performed byincubating the cells with 500 μM peptide and 5 μg/ml TS2/16 (beta 1activating antibody (IBGRL) at 0° C. for 15 minutes before theiraddition to the ICAM-4Fc coated plates.

The F and the G strand peptides (SEQ ID NOs: 10 and 11, respectively)inhibit adhesion whereas the strand A and D (SEQ ID NOs: 9 and 13,respectively) peptides had no effect. This suggests, along with the dataalready provided of the peptide inhibition of HEL cell—ICAM-4 adhesion(see Example 1), that the area of interaction with α4β1 on ICAM-4 liesin the F and G strands of domain 1. Therefore, the peptides of SEQ IDNOs: 9, 10 and 11 are useful tools allowing blocking of further ICAM-4integrin interactions that are important in erythropoiesis and in thepathology of sickle cell disease, for example.

EXAMPLE 3

Peptide and Antibody Inhibition of Neutrophil Adhesion to ICAM-4

ICAM-4 binds to platelet αIIbβ3 and the β2 integrins. These interactionsmay be part of the process whereby red cells participate in normalhemostatic processes and may also be relevant to thrombotic conditionssuch as deep vein thrombosis and vaso-occlusion in sickle cell disease.Indeed, it has recently been shown that during sickle cell crisisneutrophils that express β2 integrins, αLβ2 and αMβ2, bind not onlyinflamed endothelium but also adhere to erythrocytes. Since ICAM-4 is alikely, perhaps the only, candidate for mediating this erythrocyteadhesion with β2 integrins, we have assayed the in vitro adhesion ofneutrophils to ICAM-4.

Utilising blocking β integrin subunit antibodies and our blocking ICAM-4peptides (i.e., SEQ ID NOs: 9, 10, 11 and 13—see Example 1) in amicroplate neutrophil adhesion assay. Neutrophils were purified frombuffy coats (obtained from the National Blood Service, Bristol, UK) asdescribed in Henderson et al. (1987, Biochem. J. 246: 325-329). Celladhesion assays were performed as described in Example 1 above.Immulon-4 96 well plates (Dynes Technologies, Billingshurst, UK) werecoated with 1 μg/well protein A (Sigma, Poole, UK) for 24 hours at 4°C., washed three times with PBS and coated with 0.125 μg/well Fc fusionICAM-4 protein (ICAM-4Fc) for 18 hours at 4° C. before blocking with0.4% BSA PBS for 2 hours at 22° C. Neutrophils were labelled with 10μg/ml 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluoresceinacetoxymethyl ester (Sigma, Poole, UK) in assay buffer (Iscoves modifiedEagle medium, 2 mM EGTA, 0.1% BSA for 15 minutes at 37° C. Neutrophilswere washed with assay buffer containing 2 mM Mn²⁺. Neutrophils, at5×10⁵ cells per well, were added to the ICAM-4Fc-coated plates for 10minutes at 37° C., prior to being cyclically read on a fluorescencemicroplate reader (excitation 485 nm, emission 530 nm) and washed inassay buffer. The percentage of bound cells was calculated after eachwash. Peptide and antibody inhibition was performed by incubating thecells with 500 μM peptide and 25 μg/ml antibody at 0° C. for 15 minutesbefore their addition to the ICAM-4 Fc coated plates. Antibodies usedwere β₁ Mab13 (Yamada UK), β₂ TS1/18 (EBGRL) and β₃ PM6/13 (Serotec,UK).

We show in FIG. 20 that neutrophil—ICAM-4 adhesion is mediated by β2integrins (αLβ2 and αMβ2) and that it is likely to involve aninteraction with the G and F strands of domain 1 of ICAM-4 as opposed tothe A strand. These results are consistent with a previous site directedmutagenesis study of ICAM-4 that identified 8 residues T91, R52, E151,T154, W93, L80, R97 and W77 as important for adhesion to the β2integrins (Hermand et al., 2000, supra). All of these residues areinvolved in binding αMβ2 but only W93, L80, R97 and W77 comprise theαLβ2 binding site. T91, W93, L80, R97 and W77 are all located on the Gand F strands of domain 1 of ICAM-4.

Neutrophils bind the endothelium and to sickle red cells and thus arelikely to be important in the blockage of capillaries (vaso-occlusion)in sickle cell disease. Example 3 shows that the adhesion betweenneutrophils and ICAM-4 is β2 integrin mediated and that the peptides ofSEQ ID NO: 10 and 11 inhibit this interaction. This means thatantagonists to ICAM-4 such as SEQ ID NO: 10 and 11 could be used toaffect (for example, inhibit) hemostatic processes as well as thromboticconditions such as deep vein thrombosis and vaso-occlusion in sicklecell disease.

1. An epitope for binding integrins, comprising strands A and G ofdomain 1 of ICAM4 (SEQ ID NO: 1), in which the A strand (SEQ ID NO: 2)is defined by amino acid residues 17 to 27 of ICAM-4 and the G strand(SEQ ID NO: 3) is defined by amino acid residues 90 to 100 of ICAM-4, ora functional homologue of the epitope.
 2. The epitope according to claim1, defined by amino acid residues F18, W19, V20 on the A strand ofICAM-4 and amino acid residues R92, A94, T95, S96 and R97 on the Gstrand of ICAM-4.
 3. The epitope according to claim 1, modified in thatthe A strand is replaced by strand F on domain 1 of ICAM-4, in which theF strand (SEQ ID NO: 4) is defined by amino acid residues 77 to 87 ofICAM-4.
 4. The epitope according to claim 3, defined by amino acidresidues W77 and L80 on the F strand of ICAM-4 and amino acid residuesR92, A94, T95, S96 and R97 on the G strand of ICAM-4.
 5. The epitopeaccording to claim 1, further defined by amino acid residues W66 on theE strand of domain 1 of ICAM-4 and K118 on the B strand of domain 2 ofICAM-4, in which the E strand (SEQ ID NO: 5) is defined by amino acidresidues 160 to 170 of ICAM-4 and the B strand (SEQ ID NO: 6) is definedby amino acid residues 116 to 126 of ICAM-4.
 6. The epitope according toclaim 1, further defined by amino acid residues N160, V161 and T162 onthe E strand of ICAM-4.
 7. The epitope according to claim 1, in whichthe integrins are α_(v) integrins (for example, as found on HT1080cells), α₄β1 (also known as VLA-4; for example, as found on HEL cellsand erythroblasts), or a5P1 (for example, as found on erythroblasts). 8.A footprint domain for binding integrins, comprising a first epitope asdefined in claim 1 and a second epitope comprising the C and F strandsof domain 1 of ICAM-4 and the CE loop of domain 2 of ICAM-4, in whichthe C strand (SEQ ID NO: 7) is defined by amino acid residues 47 to 54of ICAM-4, the F strand (SEQ ID NO: 4) is defined by amino acid residues77 to 87 of ICAM-4 and the CE loop (SEQ ID NO: 8) is defined by aminoacid residues 150 to 158 of ICAM-4, or a functional homologue of thefootprint domain.
 9. The footprint domain according to claim 8, in whichthe second epitope is defined by amino acid residues R52 on the C strandof ICAM-4, W77 and L80 on the F strand of ICAM-4, T91, W93 and R97 onthe G strand of ICAM-4, and El51 and T154 on the C′-E loop of ICAM-4.10. The footprint domain according to of claim 8, in which the integrinligands are ax integrins (for example, as found on HT1080 cells), VLA-4(for example, as found on HEL cells) and/or the β₂-family of integrins(such as Mac-1, for example, as found on leucocytes and on neutrophils,and/or LFA-1), including αLβ2 (for example, as found on neutrophils).11. An antagonist of the epitope of claims
 1. 12. An antagonist of aligand for the epitope of claims
 1. 13. The antagonist of claim 12,having or consisting essentially of three, four, five, six, seven,eight, nine or more amino acid residues of the A, C, F or G strands orthe CE loop of ICAM-4, or a functional homologue thereof.
 14. Theantagonist of claim 12, in which the antagonist has or consistsessentially of the amino acid sequence according to SEQ ID NO: 9, SEQ IDNO: 10 or SEQ ID NO:
 11. 15. A method of antagonising the epitope ofclaims 1, comprising the step of contacting the epitope with anantagonist of the epitope for binding integrins.
 16. A method ofantagonising a ligand of the epitope of claims 1, comprising the step ofcontacting the ligand with an antagonist of a ligand of the epitope forbinding integrins.
 17. A method of treating a disease using theantagonist of claim
 11. 18. The method according to claim 17, in whichthe disease involves ICAM-4.
 19. A method of making a medicament for thetreatment of a disease comprising the antagonist according to claim 11,wherein the disease involves ICAM-4.
 20. The method according to claim17, in which disease is characterised by increased levels of ICAM-4binding.
 21. The method according to claim 17, in which the disease ischaracterised by decreased levels of ICAM-4 binding.
 22. The methodaccording to claim 17, in which the disease is sickle cell disease, deepvein thrombosis (DVT), malaria, heart disease, vascular complications,diabetes, β-thalassemia, or a thrombotic complication of haematologicaldiseases.
 23. An isolated nucleotide encoding the epitope defined inclaims 1 or the an antagonist thereof.
 24. The isolated nucleotide ofclaim 23, having a sequence defined within the sequence of SEQ ID NO:12.